Promoter, promoter control elements, and combinations, and uses thereof

ABSTRACT

The present invention is directed to promoter sequences and promoter control elements, polynucleotide constructs comprising the promoters and control elements, and methods of identifying the promoters, control elements, or fragments thereof. The invention further relates to the use of the present promoters or promoter control elements to modulate transcript levels in plants, and plants containing such promoters or promoter control elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending application Ser. No.14/476,566 filed on Sep. 3, 2014, which application is a Divisional ofapplication Ser. No. 12/865,719 filed on Jul. 30, 2010 (now abandoned),which is a National Phase of PCT International Application No.PCT/US09/32485 filed on Jan. 29, 2009, which claims priority to U.S.provisional application Ser. No. 61/025,697, filed on Feb. 1, 2008. Allof the above applications are hereby expressly incorporated by referenceinto the present application.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING OR TABLE

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying file, named2014_09_03_Sequence_Listing_2750-1720PUS3.txt was created on May 20,2013 and is 70 KB. The file can be accessed using Microsoft Word on acomputer that uses Windows OS.

FIELD OF THE INVENTION

The present invention relates to promoters and promoter control elementsthat are useful for modulating transcription of a desiredpolynucleotide. Such promoters and promoter control elements can beincluded in polynucleotide constructs, expression cassettes, vectors, orinserted into the chromosome or as an exogenous element, to modulate invivo and in vitro transcription of a polynucleotide. Host cells,including plant cells, and organisms, such as regenerated plantstherefrom, with desired traits or characteristics using polynucleotidescomprising the promoters and promoter control elements of the presentinvention are also a part of the invention.

BACKGROUND OF THE INVENTION

This invention relates to promoter sequences and promoter controlelement sequences which are useful for the transcription ofpolynucleotides in a host cell or transformed host organism.

The introduction of genes into plants has resulted in the development ofplants having new and useful phenotypes such as pathogen resistance,higher levels of healthier types of oils, novel production of healthfulcomponents such as beta-carotene synthesis in rice. An introduced geneis generally a chimeric gene composed of the coding region that confersthe desired trait and regulatory sequences. One regulatory sequence isthe promoter, which is located 5′ to the coding region. This sequence isinvolved in regulating the pattern of expression of a coding region 3′thereof. The promoter sequence binds RNA polymerase complex as well asone or more transcription factors that are involved in producing the RNAtranscript of the coding region.

The promoter region of a gene used in plant transformation is most oftenderived from a different source than is the coding region. It may befrom a different gene of the same species of plant, from a differentspecies of plant, from a plant virus, an algae species, a fungalspecies, or it may be a composite of different natural and/or syntheticsequences. Properties of the promoter sequence generally determine thepattern of expression for the coding region that is operably linked tothe promoter. Promoters with different characteristics of expressionhave been described. The promoter may confer broad expression as in thecase of the widely-used cauliflower mosaic virus (CaMV) 35S promoter.The promoter may confer tissue-specific expression as in the case of theseed-specific phaseolin promoter. The promoter may confer a pattern fordevelopmental changes in expression. The promoter may be induced by anapplied chemical compound, or by an environmental condition applied tothe plant.

The promoter that is used to regulate a particular coding region isdetermined by the desired expression pattern for that coding region,which itself is determined by the desired resulting phenotype in theplant. For example, herbicide resistance is desired throughout the plantso the 35S promoter is appropriate for expression of anherbicide-resistance gene. A seed-specific promoter is appropriate forchanging the oil content of soybean seed. An endosperm-specific promoteris appropriate for changing the starch composition of corn seed. Aroot-specific promoter can be important for improving water or nutrientup-take in a plant. Control of expression of an introduced gene by thepromoter is important because it is sometimes detrimental to haveexpression of an introduced gene in non-target tissues. For example, agene which induces cell death can be expressed in male and/or femalegamete cells in connection with bioconfinement.

One of the primary goals of biotechnology is to obtain organisms, suchas plants, mammals, yeast, and prokaryotes having particular desiredcharacteristics or traits. Examples of these characteristics or traitsabound and may include, for example, in plants, virus resistance, insectresistance, herbicide resistance, enhanced stability or additionalnutritional value. Recent advances in genetic engineering have enabledresearchers in the field to incorporate polynucleotide sequences intohost cells to obtain the desired qualities in the organism of choice.This technology permits one or more polynucleotides from a sourcedifferent than the organism of choice to be transcribed by the organismof choice. If desired, the transcription and/or translation of these newpolynucleotides can be modulated in the organism to exhibit a desiredcharacteristic or trait. Alternatively, new patterns of transcriptionand/or translation of polynucleotides endogenous to the organism can beproduced.

SUMMARY OF THE INVENTION

The present invention is directed to isolated polynucleotide sequencesthat comprise promoters and promoter control elements from plants,especially Arabidopsis thaliana, and other promoters and promotercontrol elements functional in plants.

It is an object of the present invention to provide isolatedpolynucleotides that are promoter or promoter control sequences. Thesepromoter sequences comprise, for example,

-   -   (1) a polynucleotide having a nucleotide sequence according to        any one of SEQ. ID. Nos. 1-26 or residues 601-1000 of SEQ ID NO:        26;    -   (2) a polynucleotide having a nucleotide sequence having at        least 80% sequence identity to a sequence according to SEQ. ID.        Nos. 1-26 or residues 601-1000 of SEQ ID NO: 26; and    -   (3) a polynucleotide having a nucleotide sequence which        hybridizes to a sequence according to SEQ. ID. Nos. 1-26 or        nucleic acid residues 601-1000 of SEQ ID NO: 26 under a        condition establishing a Tm-5° C.

Promoter or promoter control element sequences of the present inventionare capable of modulating preferential transcription.

In another embodiment, the present promoter control elements are capableof serving as or fulfilling the function, for example, as a corepromoter, a TATA box, a polymerase binding site, an initiator site, atranscription binding site, an enhancer, an inverted repeat, a locuscontrol region, and/or a scaffold/matrix attachment region.

It is yet another object of the present invention to provide apolynucleotide that includes at least a first and a second promotercontrol element. The first promoter control element is a promotercontrol element sequence as discussed above, and the second promotercontrol element is heterologous to the first control element; wherein,the first and second control elements are operably linked. Suchpromoters may modulate transcript levels preferentially in a particulartissue or under particular conditions.

In another embodiment, the present isolated polynucleotide comprises apromoter or a promoter control element as described above, wherein thepromoter or promoter control element is operably linked to apolynucleotide to be transcribed.

In another embodiment of the present invention, the promoter andpromoter control elements of the instant invention are operably linkedto a heterologous polynucleotide that is a regulatory sequence.

It is another object of the present invention to provide a host cellcomprising an isolated polynucleotide or vector as described above orfragment thereof. Host cells include, for instance, bacterial, yeast,insect, mammalian, fungus, algae, and plant. The host cell can comprisea promoter or promoter control element exogenous to the genome. Such apromoter can modulate transcription in cis- and in trans-.

In yet another embodiment, the host cell is a plant cell capable ofregenerating into a plant.

It is yet another embodiment of the present invention to provide a plantcomprising an isolated polynucleotide or vector described above.

It is another object of the present invention to provide a method ofmodulating transcription in a sample that contains either a cell-freesystem of transcription or host cell. This method comprises providing apolynucleotide or vector according to the present invention as describedabove, and contacting the sample of the polynucleotide or vector withconditions that permit transcription.

In another embodiment of the present method, the polynucleotide orvector preferentially modulates, depending upon the function of theparticular promoter, constitutive transcription, stress inducedtranscription, light induced transcription, dark induced transcription,leaf transcription, root transcription, stem or shoot transcription,silique or fruit transcription, callus transcription, rhizometranscription, stem node transcription, gamete tissue transcription,flower transcription, immature bud and inflorescence specifictranscription, senescing induced transcription, germinationtranscription and/or drought transcription.

One embodiment of the invention is directed to an isolated nucleic acidmolecule having promoter activity comprising a nucleotide sequenceselected from the group consisting of:

-   -   a. a nucleotide sequence according to any one of SEQ ID NOs.        1-26;    -   b. a nucleotide sequence of nucleic acid residues 601-1000 of        SEQ ID NO: 26;    -   c. a nucleotide sequence comprising a functional fragment of (a)        or (b), wherein said fragment has promoter activity,        and wherein said isolated nucleic acid molecule is not SEQ ID        NO: 5.

Another embodiment of the invention is directed to an isolated nucleicacid molecule comprising a nucleotide sequence that shows at least 80percent sequence identity to any one of SEQ ID NOs: 1-26 or nucleic acidresidues 601-1000 of SEQ ID NO: 26, wherein said nucleic acid moleculecomprises a regulatory region that directs transcription of an operablylinked heterologous polynucleotide, and wherein said isolated nucleicacid molecule is not SEQ ID NO: 5.

In another embodiment of the invention the isolated nucleic acidmolecule shows at least 85 percent sequence identity to any one of SEQID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment of the invention the isolated nucleic acidmolecule has at least 90 percent sequence identity to any one of SEQ IDNOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment the isolated nucleic acid molecule comprises atleast one member selected from the group consisting of a promoter, anenhancer and an intron.

In a further embodiment of the invention, the isolated nucleic acidmolecule consists of any one of SEQ ID NOs: 1-4, 6-26 and the nucleicacid residues 601-1000 of SEQ ID NO: 26.

Another embodiment of the invention is directed to a vector constructcomprising:

-   -   a. a first nucleic acid molecule as described above; and    -   b. a transcribable polynucleotide molecule,        wherein said first nucleic acid molecule and said transcribable        polynucleotide molecule are heterologous to each other and are        operably linked.

In another embodiment of the invention, the first nucleic acid moleculeconsists of the nucleic acid molecule set forth in any one of SEQ IDNOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment of the invention, the transcribable polynucleotidemolecule encodes a polypeptide.

In another embodiment of the invention, the transcribable polynucleotidemolecule is operably linked to said first nucleic acid molecule in thesense orientation.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an RNA molecule that expresses thepolypeptide encoded by transcribable polynucleotide molecule.

In another embodiment of the invention, the transcribable polynucleotidemolecule is operably linked to said first nucleic acid molecule in theantisense orientation.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an antisense RNA molecule.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an interfering RNA against an endogenousgene.

In another embodiment of the invention, the transcribable polynucleotidemolecule encodes a polypeptide of agronomic interest.

Another embodiment of the invention is directed to a plant or plant cellcomprising:

-   -   a. the nucleic acid molecule described above that is operably        linked to a heterologous polynucleotide, or    -   b. the vector construct described above.

Another embodiment of the invention is directed to a plant or plant cellstably transformed with the vector construct described above.

Another embodiment of the invention is directed to a seed of a plant asdescribed above.

Another embodiment of the invention is directed to a method of directingtranscription by combining, in an environment suitable fortranscription:

-   -   a. a first nucleic acid molecule as described above; and    -   b. a transcribable polynucleotide molecule;        wherein said first nucleic acid molecule and said transcribable        polynucleotide molecule are heterologous to each other and        operably linked.

Another embodiment of the invention is directed to a method ofexpressing an exogenous coding region in a plant comprising:

-   -   a. transforming a plant cell with a vector as described above,    -   b. regenerating a stably transformed plant from the transformed        plant cell of step (a); and    -   c. selecting plants containing a transformed plant cell,        wherein expression of the transcribable polynucleotide molecule        results in production of a polypeptide encoded by said        transcribable polynucleotide molecule.

Another embodiment of the invention is directed to a method of alteringthe expression of a gene in a plant comprising:

-   -   a. transforming a plant cell with the nucleic acid molecule as        described above that is operably linked to a heterologous        polynucleotide, and    -   b. regenerating stably transformed plants from said transformed        plant cell.

Another embodiment of the invention is directed to a plant preparedaccording to the method described above.

Another embodiment of the invention is directed to a seed from the plantdescribed above.

Another embodiment of the invention is directed to a method of producinga transgenic plant, said method comprising;

-   -   a. introducing into a plant cell:        -   (i) an isolated polynucleotide comprising the nucleic acid            as described above that is operably linked to a heterologous            polynucleotide, or        -   (ii) the vector as described above; and    -   b. growing a plant from said plant cell.

Other and further objects of the present invention will be made clear orbecome apparent from the following description.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

The Tables consist of the Expression Reports for some of the promotersof the invention providing the nucleotide sequence for each promoter anddetails for expression driven by each of the nucleic acid promotersequences as observed in transgenic plants. The results are presented assummaries of the spatial expression, which provides information as togross and/or specific expression in various plant organs and tissues.The observed expression pattern is also presented, which gives detailsof expression during different generations or different developmentalstages within a generation. Additional information is provided regardingthe source organism of the promoter, and the vector and marker genesused for the construct. The following symbols are used consistentlythroughout the Tables:

-   -   T1: First generation transformant    -   T2: Second generation transformant    -   T3: Third generation transformant    -   (L): low expression level    -   (M): medium expression level    -   (H): high expression level

Each row of the table begins with heading of the data to be found in thesection. The following provides a description of the data to be found ineach section:

Heading in Tables Description 1. Promoter Expression Report # Identifiesthe particular promoter by its construct ID. 2. Promoter tested in:Identifies the organism in which the promoter- marker vector was tested.3. Spatial expression summary: Identifies the specific parts of theplant where various levels of GFP expression are observed. Expressionlevels are noted as either low (L), medium (M), or high (H). 4. Observedexpression pattern: Provides a general explanation of where GFPexpression in different generations of plants was observed. 5. Sourcepromoter organism: Identifies the plant species from which the promoterwas derived. 6. Vector: Identifies the vector used into which a promoterwas cloned. 7. Marker type: Identifies the type of marker linked to thepromoter. The marker is used to determine patterns of gene expression inplant tissue. 8. Generation screened: T1 Mature Identified the plantgeneration(s) used in the T2 Seedling T2 Mature T3 screening process. T1plants are those plants Seedling subjected to the transformation eventwhile the T2 generation plants are from the seeds collected from the T1plants and T3 plants are from the seeds of T2 plants. 9. Inductionscompleted: Provides summary of experiment schedule. 10. T1 Mature PlantExpression: Identifies plant tissues that were observed for possibleexpression, and identifies (H, M or L) level of observed expression. 11.T2 Seedling Expression: Identifies plant tissues that were observed forpossible expression, and identifies (H, M or L) level of observedexpression. 12. T2 Mature Plant Expression: Identifies plant tissuesthat were observed for possible expression, and identifies (H, M or L)level of observed expression. 13. Utility Provides a description of theutility of the sequence, including a trait area and, in some instances,a sub-trait area. 14. Construct Identifies the promoter by its constructID and Promoter Candidate I.D. internal candidate number, and cDNAnumber cDNA I.D. 15. Lines/Events expressing: Identifies the line/eventnumbers that expressed under the promoter.Some promoter reports describe additional experiments and results withthe particular promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a vector pNewbin4-HAP1-GFP thatis useful to insert promoters of the invention into a plant. Thedefinitions of the abbreviations used in the vector map are as follows:

-   Ori—the origin of replication used by an E. coli host-   RB—sequence for the right border of the T-DNA from pMOG800-   BstXI—restriction enzyme cleavage site used for cloning-   HAP1VP16—coding sequence for a fusion protein of the HAP1 and VP16    activation domains-   NOS—terminator region from the nopaline synthase gene-   HAP1UAS—the upstream activating sequence for HAP1-   5ERGFP—the green fluorescent protein gene that has been optimized    for localization to the endoplasmic reticulum-   OCS2—the terminator sequence from the octopine synthase 2 gene-   OCS—the terminator sequence from the octopine synthase gene-   p28716 (a.k.a. 28716 short)—promoter used to drive expression of the    PAT (BAR) gene-   PAT (BAR)—a marker gene conferring herbicide resistance-   LB—sequence for the left border of the T-DNA from pMOG800-   Spec—a marker gene conferring spectinomycin resistance-   TrfA—transcription repression factor gene-   RK2-OriV—origin of replication for Agrobacterium

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The invention disclosed herein provides promoters capable of driving theexpression of an operably linked transgene. The design, construction,and use of these promoters is one object of this invention. The promotersequences, SEQ ID NOs: 1-26 and residues 601-1000 of SEQ ID NO: 26, arecapable of transcribing operably linked nucleic acid molecules inparticular plant tissues/organs or during particular plant growthstages, and therefore can selectively regulate expression of transgenesin these tissues/organs or at these times of plant development.

1. Definitions

Chimeric: The term “chimeric” is used to describe polynucleotides orgenes, or constructs wherein at least two of the elements of thepolynucleotide or gene or construct, such as the promoter and thepolynucleotide to be transcribed and/or other regulatory sequencesand/or filler sequences and/or complements thereof, are heterologous toeach other.

Broadly Expressing Promoter: Promoters referred to herein as “broadlyexpressing promoters” actively promote transcription under most, but notnecessarily all, environmental conditions and states of development orcell differentiation. Examples of broadly expressing promoters includethe cauliflower mosaic virus (CaMV) 35S transcript initiation region andthe 1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens,and other transcription initiation regions from various plant genes,such as the maize ubiquitin-1 promoter, known to those of skill.

Domain: Domains are fingerprints or signatures that can be used tocharacterize protein families and/or parts of proteins. Suchfingerprints or signatures can comprise conserved (1) primary sequence,(2) secondary structure, and/or (3) three-dimensional conformation. Asimilar analysis can be applied to polynucleotides. Generally, eachdomain has been associated with either a conserved primary sequence or asequence motif. Generally these conserved primary sequence motifs havebeen correlated with specific in vitro and/or in vivo activities. Adomain can be any length, including the entirety of the polynucleotideto be transcribed. Examples of domains include, without limitation, AP2,helicase, homeobox, zinc finger, etc.

Endogenous: The term “endogenous,” within the context of the currentinvention refers to any polynucleotide, polypeptide or protein sequencewhich is a natural part of a cell or organism(s) regenerated from saidcell. In the context of promoter, the term “endogenous coding region” or“endogenous cDNA” refers to the coding region that is naturally operablylinked to the promoter.

Enhancer/Suppressor: An “enhancer” is a DNA regulatory element that canincrease the steady state level of a transcript, usually by increasingthe rate of transcription initiation. Enhancers usually exert theireffect regardless of the distance, upstream or downstream location, ororientation of the enhancer relative to the start site of transcription.In contrast, a “suppressor” is a corresponding DNA regulatory elementthat decreases the steady state level of a transcript, again usually byaffecting the rate of transcription initiation. The essential activityof enhancer and suppressor elements is to bind a protein factor(s). Suchbinding can be assayed, for example, by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in an in vitro transcription extract.

Exogenous: As referred to within, “exogenous” is any polynucleotide,polypeptide or protein sequence, whether chimeric or not, that isintroduced into the genome of a host cell or organism regenerated fromsaid host cell by any means other than by a sexual cross. Examples ofmeans by which this can be accomplished are described below, and includeAgrobacterium-mediated transformation (of dicots—e.g. Salomon et al.(1984) EMBO J. 3:141; Herrera-Estrella et al. (1983) EMBO J. 2:987; ofmonocots, representative papers are those by Escudero et al. (1996)Plant J. 10:355), Ishida et al. (1996) Nature Biotech 14:745, May et al.(1995) Bio/Technology 13:486), biolistic methods (Armaleo et al. (1990)Current Genetics 17:97), electroporation, in planta techniques, and thelike. Such a plant containing the exogenous nucleic acid is referred tohere as a T₀ for the primary transgenic plant and T₁ for the firstgeneration. The term “exogenous” as used herein is also intended toencompass inserting a naturally found element into a non-naturally foundlocation.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Homologous: In the current invention, a “homologous” polynucleotiderefers to a polynucleotide that shares sequence similarity with thepolynucleotide of interest. This similarity may be in only a fragment ofthe sequence and often represents a functional domain such as, examplesincluding, without limitation, a DNA binding domain or a domain withtyrosine kinase activity. The functional activities of homologouspolynucleotides are not necessarily the same.

Inducible Promoter: An “inducible promoter” in the context of thecurrent invention refers to a promoter, the activity of which isinfluenced by certain conditions, such as light, temperature, chemicalconcentration, protein concentration, conditions in an organism, cell,or organelle, etc. A typical example of an inducible promoter, which canbe utilized with the polynucleotides of the present invention, isPARSK1, the promoter from an Arabidopsis gene encoding aserine-threonine kinase enzyme, and which promoter is induced bydehydration, abscisic acid and sodium chloride (Wang and Goodman (1995)Plant J. 8:37). Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions,elevated temperature, the presence or absence of a nutrient or otherchemical compound or the presence of light.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene or coding region from a different plantspecies or from a non-plant organism.

Modulate Transcription Level: As used herein, the phrase “modulatetranscription” describes the biological activity of a promoter sequenceor promoter control element. Such modulation includes, withoutlimitation, up- and down-regulation of initiation of transcription, rateof transcription, and/or transcription levels.

Operable Linkage: An “operable linkage” is a linkage in which a promotersequence or promoter control element is connected to a polynucleotidesequence (or sequences) in such a way as to place transcription of thepolynucleotide sequence under the influence or control of the promoteror promoter control element. Two DNA sequences (such as a polynucleotideto be transcribed and a promoter sequence linked to the 5′ end of thepolynucleotide to be transcribed) are said to be operably linked ifinduction of promoter function results in the transcription of mRNAencoding the polynucleotide and if the nature of the linkage between thetwo DNA sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promotersequence to direct the expression of the protein, antisense RNA, RNAi orribozyme, or (3) interfere with the ability of the DNA template to betranscribed. Thus, a promoter sequence would be operably linked to apolynucleotide sequence if the promoter was capable of effectingtranscription of that polynucleotide sequence.

Percentage of sequence identity As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence, e.g., SEQ ID NOs:1-26, and a subject sequence. A subjectsequence typically has a length that is from about 80 percent to 250percent of the length of the query sequence, e.g., 82, 85, 87, 89, 90,93, 95, 97, 99, 100, 105, 110, 115, or 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, or 250 percent of the length of thequery sequence. A query nucleic acid or amino acid sequence is alignedto one or more subject nucleic acid or amino acid sequences using thecomputer program ClustalW (version 1.83, default parameters), whichallows alignments of nucleic acid or protein sequences to be carried outacross their entire length (global alignment). Chenna et al. (2003)Nucleic Acids Res. 31(13):3497-500.

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For an alignment of multiple nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

To determine a percent identity of a subject polypeptide or nucleic acidsequence to a query sequence, the sequences are aligned using Clustal W,the number of identical matches in the alignment is divided by thelength of the query sequence, and the result is multiplied by 100. Theoutput is the percent identity of the subject sequence with respect tothe query sequence. It is noted that the percent identity value can berounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and78.19 are rounded up to 78.2.

Plant Promoter: A “plant promoter” is a promoter capable of initiatingtranscription in plant cells and can modulate transcription of apolynucleotide. Such promoters need not be of plant origin. For example,promoters derived from plant viruses, such as the CaMV35S promoter orfrom Agrobacterium tumefaciens such as the T-DNA promoters, can be plantpromoters. A typical example of a plant promoter of plant origin is themaize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.

Plant Tissue: The term “plant tissue” includes differentiated andundifferentiated tissues or plants, including but not limited to roots,stems, shoots, rhizomes, cotyledons, epicotyl, hypocotyl, leaves,pollen, seeds, gall tissue and various forms of cells in culture such assingle cells, protoplast, embryos, and callus tissue. The plant tissuemay be in plants or in organ, tissue or cell culture.

Preferential Transcription: “Preferential transcription” is defined astranscription that occurs in a particular pattern of cell types ordevelopmental times or in response to specific stimuli or combinationthereof. Non-limitive examples of preferential transcription include:high transcript levels of a desired sequence in root tissues; detectabletranscript levels of a desired sequence in certain cell types duringembryogenesis; and low transcript levels of a desired sequence underdrought conditions. Such preferential transcription can be determined bymeasuring initiation, rate, and/or levels of transcription.

Promoter: A “promoter” is a DNA sequence that directs the transcriptionof a polynucleotide. Typically a promoter is located in the 5′ region ofa polynucleotide to be transcribed, proximal to the transcriptionalstart site of such polynucleotide. More typically, promoters are definedas the region upstream of the first exon; more typically, as a regionupstream of the first of multiple transcription start sites; moretypically, as the region downstream of the preceding gene and upstreamof the first of multiple transcription start sites; more typically, theregion downstream of the polyA signal and upstream of the first ofmultiple transcription start sites; even more typically, about 3,000nucleotides upstream of the ATG of the first exon; even more typically,2,000 nucleotides upstream of the first of multiple transcription startsites. The promoters of the invention comprise at least a core promoteras defined above. Frequently promoters are capable of directingtranscription of genes located on each of the complementary DNA strandsthat are 3′ to the promoter. Stated differently, many promoters exhibitbidirectionality and can direct transcription of a downstream gene whenpresent in either orientation (i.e. 5′ to 3′ or 3′ to 5′ relative to thecoding region of the gene). Additionally, the promoter may also includeat least one control element such as an upstream element. Such elementsinclude UARs and optionally, other DNA sequences that affecttranscription of a polynucleotide such as a synthetic upstream element.

Promoter Control Element: The term “promoter control element” as usedherein describes elements that influence the activity of the promoter.Promoter control elements include transcriptional regulatory sequencedeterminants such as, but not limited to, enhancers, scaffold/matrixattachment regions, TATA boxes, transcription start locus controlregions, UARs, URRs, other transcription factor binding sites andinverted repeats.

Public sequence: The term “public sequence,” as used in the context ofthe instant application, refers to any sequence that has been depositedin a publicly accessible database prior to the filing date of thepresent application. This term encompasses both amino acid andnucleotide sequences. Such sequences are publicly accessible, forexample, on the BLAST databases on the NCBI FTP web site (accessible viathe internet). The database at the NCBI FTP site utilizes “gi” numbersassigned by NCBI as a unique identifier for each sequence in thedatabases, thereby providing a non-redundant database for sequence fromvarious databases, including GenBank, EMBL, DBBJ (DNA Database of Japan)and PDB (Brookhaven Protein Data Bank).

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns.

The nucleic acid sequence set forth in SEQ ID NOs:1-26 are examples ofregulatory regions provided herein. However, a regulatory region canhave a nucleotide sequence that deviates from that set forth in SEQ IDNOs:1-26, while retaining the ability to direct expression of anoperably linked nucleic acid. For example, a regulatory region having80% or greater (e.g. 85% or greater, 90% or greater 91% or greater, 92%or greater, 93% or greater, 94% or greater, 95% or greater, 96% orgreater, 97% or greater, 98% or greater, or 99% or greater) sequenceidentity to the nucleotide sequence set forth in SEQ ID NOs:1-25, or 26can direct expression of an operably linked nucleic acid.

A regulatory region can also be a fragment of SEQ ID NOs:1-25, or 26,while retaining promoter activity, i.e. the ability to direct expressionof an operably linked nucleic acid. Additional examples of regulatoryregions are identified in the Sequence Listing.

Regulatory regions can be classified in two categories, promoters andother regulatory regions.

Regulatory Sequence: The term “regulatory sequence,” as used in thecurrent invention, refers to any nucleotide sequence that influencestranscription or translation initiation and rate, or stability and/ormobility of a transcript or polypeptide product. Regulatory sequencesinclude, but are not limited to, promoter sequences, enhancer sequences,response elements, protein recognition sites, inducible elements,promoter control elements, protein binding sequences, 5′ and 3′untranslated regions (UTRs), transcriptional start sites, terminationsequences, polyadenylation sequences, introns, certain sequences withinamino acid coding sequences such as secretory signals, protease cleavagesites, etc.

A 5′ untranslated region (5′ UTR) of a gene is generally defined as apolynucleotide segment between the transcription start site (TSS) andthe coding sequence start site (ATG codon) of a messenger RNA or cDNA.Alternately, 5′ UTR can be synthetically produced or manipulated DNAelements. A “plant 5′UTR” can be a native or non-native 5′UTR that isfunctional in plant cells. A 5′ UTR can be used as a 5′ regulatoryelement for modulating expression of an operably linked transcribablepolynucleotide molecule. For example, 5′ UTRs derived from heat shockprotein genes have been demonstrated to enhance gene expression inplants (see for example, U.S. Pat. Nos. 5,659,122 and 5,362,865, all ofwhich are incorporated herein by reference). Examples of 5′UTRs includethose shown in SEQ ID NOs: 1-4, 6-10, 13-26.

Specific Promoters: In the context of the current invention, “specificpromoters” refers to a subset of promoters that have a high preferencefor modulating transcript levels in a specific tissue, or organ or celland/or at a specific time during development of an organism. By “highpreference” is meant at least 3-fold, preferably 5-fold, more preferablyat least 10-fold still more preferably at least 20-fold, 50-fold or100-fold increase in transcript levels under the specific condition overthe transcription under any other reference condition considered.Typical examples of temporal and/or tissue or organ specific promotersof plant origin that can be used with the polynucleotides of the presentinvention, are: PTA29, a promoter which is capable of driving genetranscription specifically in tapetum and only during anther development(Koltonow et al. (1990) Plant Cell 2:1201; RCc2 and RCc3, promoters thatdirect root-specific gene transcription in rice (Xu et al. (1995) PlantMol. Biol. 27:237; TobRB27, a root-specific promoter from tobacco(Yamamoto et al. (1991) Plant Cell 3:371). Examples of tissue-specificpromoters under developmental control include promoters that initiatetranscription only in certain tissues or organs, such as root, ovule,fruit, seeds, or flowers. Other specific promoters include those fromgenes encoding seed storage proteins or the lipid body membrane protein,oleosin. A few root-specific promoters are noted above. See also“Preferential transcription.”

A regulatory region can contain conserved regulatory motifs. Such aregulatory region can be any one of the sequences set forth in SEQ IDNOs:1-26, or a regulatory region having a nucleotide sequence thatdeviates from any one of those set forth in SEQ ID NOs:1-26, whileretaining the ability to direct expression of an operably linked nucleicacid. For example, a regulatory region can contain a CAAT box or a TATAbox. A CAAT box is a conserved nucleotide sequence involved ininitiation of transcription. A CAAT box functions as a recognition andbinding site for regulatory proteins called transcription factors. ATATA box is another conserved nucleotide sequence involved intranscription initiation. A TATA box seems to be important indetermining accurately the position at which transcription is initiated.

Other conserved regulatory motifs can be identified using methods knownin the art. For example, a regulatory region can be analyzed using thePLACE (PLAnt Cis-acting regulatory DNA Elements) Web Signal Scan programon the world wide web at dna.affrc.go.jp/PLACE/signalscan.html. See,Higo et al., Nucleic Acids Research, 27(1):297-300 (1999); andPrestridge, CABIOS, 7:203-206 (1991). Examples of conserved regulatorymotifs can be found in the PLACE database on the world wide web atdna.affrc.go.jp/PLACE/. See, Higo et al., supra.

A regulatory region such as any one of SEQ ID NOs:1-26, or a regulatoryregion having a nucleotide sequence that deviates from those set forthin SEQ ID NOs:1-26, while retaining the ability to direct expression ofan operably linked nucleic acid, can contain one or more conservedregulatory motifs, which can be found in the PLACE database. Forexample, such a regulatory region can contain a −300CORE motif havingthe consensus sequence TGTAAAG (SEQ ID NO:27). See, Forde et al.,Nucleic Acids Res 13:7327-7339 (1985); Colot et al., EMBO J 6:3559-3564(1987); Thomas and Flavell, Plant Cell 2:1171-1180 (1990); Thompson etal., Plant Mol Biol 15:755-764 (1990); Vicente-Carbajosa et al., ProcNatl Acad Sci USA 94:7685-7690 (1997); Mena et al., Plant J. 16:53-62(1998); Shing, Plant Physiol 118: 1111-1120 (1998). Such a regulatoryregion can contain an ABREATCONSENSUS motif having the consensussequence YACGTGGC (SEQ ID NO:28). See, Choi et al., J Biol Chem 275:1723-1730 (2000); Kang et al., Plant Cell 14: 343-357 (2002); Oh et al.,Plant Physiology 138: 341-351 (2005); Choi et al., Plant Physiol 139:1750-1761(2005). Such a regulatory region can contain an ABREATRD22motif having the consensus sequence RYACGTGGYR (SEQ ID NO:29). See,Iwasaki et al., Mol Gen Genet 247:391-398 (1995); Bray, Trends in PlantScience 2:48-54 (1997); Busk and Pages, Plant Mol Biol 37:425-435(1998). A regulatory region can contain an ABRELATERD1 motif having theconsensus sequence ACGTG (SEQ ID NO:30). See, Simpson et al., Plant J33: 259-270 (2003); Nakashima et al., Plant Mol Biol 60:51-68 (2006). Aregulatory region can contain an ABREMOTIFAOSOSEM motif having theconsensus sequence TACGTGTC (SEQ ID NO:31). See, Hattori et al., Plant J7: 913-925 (1995); Hobo et al., Proc Natl Acad Sci USA 96: 15348-15353(1999). A regulatory region can contain an ABRERATCAL motif having theconsensus sequence MACGYGB (SEQ ID NO:32). See, Kaplan et al., PlantCell 18:2733-2748 (2006). A regulatory region can contain an ACGTCBOXmotif having the consensus sequence GACGTC (SEQ ID NO:33). See, Fosteret al., FASEB J 8:192-200 (1994); Izawa et al., Plant Cell 6:1277-1287(1994); Izawa et al., J Mol Biol 230:1131-1144 (1993). A regulatoryregion can contain an ACGTOSGLUB1 motif having the consensus sequenceGTACGTG (SEQ ID NO:34). See, Washida et al., Plant Mol Biol 40:1-12(1999); Wu et al., Plant J 23: 415-421 (2000). A regulatory region cancontain an ACGTTBOX motif having the consensus sequence AACGTT (SEQ IDNO:35). See, Foster et al., FASEB J 8:192-200 (1994). A regulatoryregion can contain an ACIIPVPAL2 motif having the consensus sequenceCCACCAACCCCC (SEQ ID NO:36). See, Patzlaff et al., Plant Mol Biol53:597-608 (2003); Hatton et al., Plant J 7:859-876 (1995);Gomez-Maldonado et al., Plant J 39:513-526 (2004). A regulatory regioncan contain an AGL2ATCONSENSUS motif having the consensus sequenceNNWNCCAWWWWTRGWWAN (SEQ ID NO:37). See, Huang et al., Plant Cell 8:81-94 (1996). A regulatory region can contain an AMYBOX2 motif havingthe consensus sequence TATCCAT (SEQ ID NO:38). See, Huang et al., PlantMol Biol 14:655-668 (1990); Hwang et al., Plant Mol Biol 36:331-341(1998). A regulatory region can contain an ANAERO1CONSENSUS motif havingthe consensus sequence AAACAAA (SEQ ID NO:39). See, Mohanty et al., AnnBot (Lond). 96: 669-681 (2005). A regulatory region can contain an ARE1motif having the consensus sequence RGTGACNNNGC (SEQ ID NO:40). See,Rushmore et al., J Biol Chem 266:11632-11639 (1991). A regulatory regioncan contain an ATHB6COREAT motif having the consensus sequence CAATTATTA(SEQ ID NO:41). See, Himmelbach et al., EMBO J 21:3029-3038 (2002). Aregulatory region can contain an AUXRETGA1GMGH3 motif having theconsensus sequence TGACGTAA (SEQ ID NO:42). See, Liu et al., Plant Cell6:645-657 (1994); Liu et al., Plant Physiol 115:397-407 (1997);Guilfoyle et al., Plant Physiol 118: 341-347 (1998). A regulatory regioncan contain a BOXIIPCCHS motif having the consensus sequence ACGTGGC(SEQ ID NO:43). See, Block et al., Proc Natl Acad Sci USA87:5387-5391(1990); Terzaghi and Cashmore, Annu Rev Plant Physiol PlantMol Biol 46:445-474 (1995); Nakashima et al., Plant Mol Biol 60: 51-68(2006). A regulatory region can contain a BOXLCOREDCPAL motif having theconsensus sequence ACCWWCC (SEQ ID NO:44). See, Meada et al., Plant MolBiol 59: 739-752.(2005). A regulatory region can contain a CACGCAATGMGH3motif having the consensus sequence CACGCAAT (SEQ ID NO:45). See,Ulmasov et al., Plant Cell 7: 1611-1623 (1995). A regulatory region cancontain a CARGATCONSENSUS motif having the consensus sequence CCWWWWWWGG(SEQ ID NO:46). See, Hepworth et al., EMBO J 21: 4327-4337 (2002);Michaels et al., Plant J 33: 867-874 (2003); Hong et al., Plant Cell15:1296-1309 (2003); Folter and Angenent, Trends Plant Sci 11:224-231(2006). A regulatory region can contain a CARGCW8GAT motif having theconsensus sequence CWWWWWWWWG (SEQ ID NO:47). See, Tang and Perry, JBiol Chem 278:28154-28159 (2003); Folter and Angenent, Trends Plant Sci11:224-231 (2006). A regulatory region can contain a CIACADIANLELHCmotif having the consensus sequence CAANNNNATC (SEQ ID NO:48). See,Piechulla et al., Plant Mol Biol 38:655-662 (1998). A regulatory regioncan contain a DPBFCOREDCDC3 motif having the consensus sequence ACACNNG(SEQ ID NO:49). See, Kim et al., Plant J 11: 1237-1251 (1997);Finkelstein and Lynch, Plant Cell 12: 599-609 (2000); Lopez-Molina andChua, Plant Cell Physiol 41: 541-547 (2000). A regulatory region cancontain a DRE2COREZMRAB17 motif having the consensus sequence ACCGAC(SEQ ID NO:50). See, Busk et al., Plant J 11: 1285-1295 (1997); Dubouzetet al., Plant J 33: 751-763 (2003); Kizis and Pages, Plant J 30: 679-689(2002). A regulatory region can contain an E2FCONSENSUS motif having theconsensus sequence WTTSSCSS (SEQ ID NO:51). See, Vandepoele et al.,Plant Physiol 139: 316-328. (2005). A regulatory region can contain anEMHVCHORD motif having the consensus sequence TGTAAAGT (SEQ ID NO:52).See, Muller and Knudsen, Plant J 4: 343-355 (1993). A regulatory regioncan contain an EVENINGAT motif having the consensus sequence AAAATATCT(SEQ ID NO:53). See, Rawat et al., Plant Mol Biol 57: 629-643 (2005) andHarmer et al., Science 290: 2110-2113 (2000). A regulatory region cancontain an GLMHVCHORD motif having the consensus sequence RTGASTCAT (SEQID NO:54). See, Albani et al., Plant Cell 9: 171-184 (1997); Muller MPlant J 4: 343-355 (1993); Onate et al., J Biol Chem 274: 9175-9182(1999). A regulatory region can contain a GT1 Consensus motif having theconsensus sequence GRWAAW (SEQ ID NO:55). See, Terzaghi and Cashmore,supra.; Villain et al., J Biol Chem 271:32593-32598 (1996); LeGourrierec et al., Plant J 18:663-668 (1999); Buchel et al., Plant MolBiol 40:387-396 (1999); Zhou, Trends in Plant Science 4:210-214 (1999).A regulatory region can contain a GT1GMSCAM4 motif having the consensussequence GAAAAA (SEQ ID NO:56). See, Park et al., Plant Physiol 135:2150-2161 (2004). A regulatory region can contain a HDZIP2ATATHB2 motifhaving the consensus sequence TAATMATTA (SEQ ID NO:57). See, Ohgishi etal., Plant J 25: 389-398 (2001). A regulatory region can contain anIBOXCORENT motif having the consensus sequence GATAAGR (SEQ ID NO:58).See, Martinez-Hernandez et al., Plant Physiol 128:1223-1233 (2002). Aregulatory region can contain an INRNTPSADB motif having the consensussequence YTCANTYY (SEQ ID NO:59). See, Nakamura et al., Plant J 29: 1-10(2002). A regulatory region can contain a LEAFYATAG motif having theconsensus sequence CCAATGT (SEQ ID NO:60). See, Kamiya et al., Plant J35: 429-441 (2003). A regulatory region can contain a LRENPCABE motifhaving the consensus sequence ACGTGGCA (SEQ ID NO:61). See, Castresanaet al., EMBO J 7:1929-1936 (1988). A regulatory region can contain aMARTBOX motif having the consensus sequence TTWTWTTWTT (SEQ ID NO:62).See, Gasser et al., Intnatl Rev Cyto 119:57-96 (1989). A regulatoryregion can contain a MYBGAHV motif having the consensus sequence TAACAAA(SEQ ID NO:63). See, Gubler et al., Plant Cell 7:1879-1891 (1995);Morita et al., FEBS Lett 423:81-85 (1998); Gubler et al., Plant J17:1-9(1999). A regulatory region can contain a MYBPLANT motif havingthe consensus sequence MACCWAMC (SEQ ID NO:64). See, Sablowski et al.,EMBO J 13:128-137 (1994); Tamagnone et al., Plant Cell 10: 135-154(1998). A regulatory region can contain a NRRBNEXTA motif having theconsensus sequence TAGTGGAT (SEQ NO:65). See, Elliott and Shirsat, PlantMol Biol 37:675-687 (1998). A regulatory region can contain an O2F3BE2S1motif having the consensus sequence TCCACGTACT (SEQ ID NO:66). See,Vincentz et al., Plant Mol Biol 34:879-889 (1997). A regulatory regioncan contain a P1BS motif having the consensus sequence GNATATNC (SEQ IDNO:67). See, Rubio et al., Genes Dev. 15: 2122-2133.(2001); Shunmann etal., J Exp Bot. 55: 855-865. (2004); Shunmann et al., Plant Physiol 136:4205-4214. (2004). A regulatory region can contain a PRECONSCRHSP70Amotif having the consensus sequence SCGAYNRNNNNNNNNNNNNNNNHD (SEQ IDNO:68). See, von Gromoff et al., Nucleic Acids Res 34:4767-4779 (2006).A regulatory region can contain a PROXBBNNAPA motif having the consensussequence CAAACACC (SEQ ID NO:69). See, Ezcurra et al., Plant Mol Biol40:699-709 (1999); Busk and Pages, supra.; Ezcurra et al., Plant J24:57-66 (2000). A regulatory region can contain a PYRIMIDINEBOXHVEPB1motif having the consensus sequence TTTTTTCC (SEQ ID NO:70). See, Cercoset al., Plant J 19: 107-118 (1999). A regulatory region can contain aRBCSCONSENSUS motif having the consensus sequence AATCCAA (SEQ IDNO:71). See, Manzara and Gruissem, Photosynth Res 16:117-139 (1988);Donald and Cashmore, EMBO J 9:1717-1726 (1990). A regulatory region cancontain a ROOTMOTIFTAPDX1 motif having the consensus sequence ATATT (SEQID NO:72). See, Elmayan and Tepfer, Transgenic Res 4:388-396 (1995). Aregulatory region can contain a RYREPEATVFLEB4 motif having theconsensus sequence CATGCATG (SEQ ID NO:73). See, Curaba et al., PlantPhysiol 136: 3660-3669. (2004); Nag et al., Plant Mol Biol 59: 821-838(2005). A regulatory region can contain a SEF1MOTIF motif having theconsensus sequence ATATTTAWW (SEQ ID NO:74). See, Allen et al., PlantCell 1:623-631 (1989); Lessard et al., Plant Mol Biol 16:397-413 (1991).A regulatory region can contain a SORLREP3AT motif having the consensussequence TGTATATAT (SEQ ID NO:75). See, Hudson and Quail, Plant Physiol133: 1605-1616 (2003). A regulatory region can contain a SURE2STPAT21motif having the consensus sequence AATACTAAT (SEQ ID NO:76). See,Grierson et al., Plant J 5:815-826 (1994). A regulatory region cancontain a SV40COREENHAN motif having the consensus sequence GTGGWWHG(SEQ ID NO:77). See, Weiher et al., Science 219:626-631 (1983); Green etal., EMBO J 6:2543-2549 (1987); Donald and Cashmore, EMBO J 9:1717-1726(1990). A regulatory region can contain a TATABOX2 motif having theconsensus sequence TATAAAT (SEQ ID NO:78). See, Shirsat et al., Mol GenGenet 215:326-331 (1989); Grace et al., Biol Chem 279:8102-8110 (2004).A regulatory region can contain a TATABOX3 motif having the consensussequence TATTAAT (SEQ ID NO:79). See, PLACE (PLAnt Cis-acting regulatoryDNA Elements) at dna.affrc.go.jp/PLCAE/signalscan.html). A regulatoryregion can contain a TATABOX4 motif having the consensus sequenceTATATAA (SEQ ID NO:80). See, Grace et al., J Biol Chem 279:8102-8110(2004). A regulatory region can contain a TATABOX5 motif having theconsensus sequence TTATTT (SEQ ID NO:81). See, Tjaden et al., PlantPhysiol 108:1109-1117 (1995). A regulatory region can contain aTATABOXOSPAL motif having the consensus sequence TATTTAA (SEQ ID NO:82).See, Zhu et al., Plant Cell 14: 795-803 (2002). A regulatory region cancontain a TELOBOXATEEF1AA1 motif having the consensus sequence AAACCCTAA(SEQ ID NO:83). See, Tremousayque et al., Plant J 20: 553-561 (1999);Axelos et al., Mol Gen Genet 219: 106-112 (1989); Welchen and Gonzalez,Plant Physiol 139: 88-100 (2005). A regulatory region can contain aTL1ATSAR motif having the consensus sequence CTGAAGAAGAA (SEQ ID NO:84).See, Wang et al., Science 308: 1036-1040 (2005). A regulatory region cancontain a UP2ATMSD motif having the consensus sequence AAACCCTA (SEQ IDNO:85). See, Tatematsu et al., Plant Physiology 138: 757-766 (2005). Aregulatory region can contain a WBBOXPCWRKY1 motif having the consensussequence TTTGACY (SEQ ID NO:86). See, Ishiguro and Nakamura, Mol GenGenet 244:563-571 (1994); Rushton et al., Plant Mol Biol 29:691-702(1995); Rushon et al., EMBO J 15:5690-5700 (1996); de Pater et al.,Nucleic Acids Res 24:4624-4631 (1996); Eulgem et al., Trends Plant Sci5: 199-206 (2000). A regulatory region can contain a XYLAT motif havingthe consensus sequence ACAAAGAA (SEQ ID NO:87). See, Ko et al., MolGenet Genomics 276:517-531 (2006).

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)-5° C. to T_(m)-10° C. Medium or moderate stringencyconditions are those providing T_(m)-20° C. to T_(m)-29° C. Lowstringency conditions are those providing a condition of T_(m)-40° C. toT_(m)-48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:T _(m)=81.5−16.6(log₁₀[Na ⁺])+0.41(% G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):T _(m)=81.5+16.6 log {[Na ⁺]/(1+0.7[Na ⁺])}+0.41(% G+C)−500/L 0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(21). The T_(m) of Equation II is affected by the nature of the hybrid:for DNA-RNA hybrids, T_(m) is 10-15° C. higher than calculated; forRNA-RNA hybrids, T_(m) is 20-25° C. higher. Because the T_(m) decreasesabout 1° C. for each 1% decrease in homology when a long probe is used(Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

TATA to start: “TATA to start” shall mean the distance, in number ofnucleotides, between the primary TATA motif and the start oftranscription.

Transgenic plant: A “transgenic plant” is a plant having one or moreplant cells that contain at least one exogenous polynucleotideintroduced by recombinant nucleic acid methods.

Translational start site: In the context of the present invention, a“translational start site” is usually an ATG or AUG in a transcript,often the first ATG or AUG. A single protein encoding transcript,however, may have multiple translational start sites.

Transcription start site: “Transcription start site” is used in thecurrent invention to describe the point at which transcription isinitiated. This point is typically located about 25 nucleotidesdownstream from a TFIID binding site, such as a TATA box. Transcriptioncan initiate at one or more sites within the gene, and a singlepolynucleotide to be transcribed may have multiple transcriptional startsites, some of which may be specific for transcription in a particularcell-type or tissue or organ. “+1” is stated relative to thetranscription start site and indicates the first nucleotide in atranscript.

Upstream Activating Region (UAR): An “Upstream Activating Region” or“UAR” is a position or orientation dependent nucleic acid element thatprimarily directs tissue, organ, cell type, or environmental regulationof transcript level, usually by affecting the rate of transcriptioninitiation. Corresponding DNA elements that have a transcriptioninhibitory effect are called herein “Upstream Repressor Regions” or“URR”s. The essential activity of these elements is to bind a proteinfactor. Such binding can be assayed by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in vitro transcription extract.

Untranslated region (UTR): A “UTR” is any contiguous series ofnucleotide bases that is transcribed, but is not translated. A 5′ UTRlies between the start site of the transcript and the translationinitiation codon and includes the +1 nucleotide. A 3′ UTR lies betweenthe translation termination codon and the end of the transcript. UTRscan have particular functions such as increasing mRNA message stabilityor translation attenuation. Examples of 3′ UTRs include, but are notlimited to polyadenylation signals and transcription terminationsequences.

2. Use of the Promoters of the Invention

The promoters and promoter control elements of this invention arecapable of modulating transcription. Such promoters and promoter controlelements can be used in combination with native or heterologous promoterfragments, control elements or other regulatory sequences to modulatetranscription and/or translation.

Specifically, promoters and control elements of the invention can beused to modulate transcription of a desired polynucleotide, whichincludes without limitation:

-   -   (i) antisense;    -   (ii) ribozymes;    -   (iii) coding sequences; or    -   (iv) fragments thereof.

The promoter also can modulate transcription in a host genome in cis- orin trans-.

In an organism, such as a plant, the promoters and promoter controlelements of the instant invention are useful to produce preferentialtranscription which results in a desired pattern of transcript levels ina particular cells, tissues, or organs, or under particular conditions.

4. Identifying and Isolating Promoter Sequences of the Invention

The promoters and promoter control elements of the present invention arepresented in the Promoter Reports of the Tables and were identified fromArabidopsis thaliana and Oryza sativa. Isolation from genomic librariesof polynucleotides comprising the sequences of the promoters andpromoter control elements of the present invention is possible usingknown techniques. For example, polymerase chain reaction (PCR) canamplify the desired polynucleotides utilizing primers designed from SEQID NOs: 1-26 or residues 601-1000 of SEQ ID NO: 26. Polynucleotidelibraries comprising genomic sequences can be constructed according toSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.(1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), for example.

Other procedures for isolating polynucleotides comprising the promotersequences of the invention include, without limitation, tail-PCR, and 5′rapid amplification of cDNA ends (RACE). See, for tail-PCR, for example,Liu et al. (1995) Plant J 8(3): 457-463; Liu et al. (1995) Genomics 25:674-681; Liu et al. (1993) Nucl. Acids Res. 21(14): 3333-3334; and Zoeet al. (1999) BioTechniques 27(2): 240-248; for RACE, see, for example,PCR Protocols: A Guide to Methods and Applications, (1990) AcademicPress, Inc.

In addition, the promoters and promoter control elements described inthe Promoter Reports in the Tables (SEQ. ID. Nos. 1-26) can bechemically synthesized according to techniques in common use. See, forexample, Beaucage et al. (1981) Tet. Lett. 22: 1859 and U.S. Pat. No.4,668,777. Such chemical oligonucleotide synthesis can be carried outusing commercially available devices, such as, Biosearch 4600 or 8600DNA synthesizer, by Applied Biosystems, a division of Perkin-ElmerCorp., Foster City, Calif., USA; and Expedite by Perceptive Biosystems,Framingham, Mass., USA.

Included in the present invention are promoters exhibiting nucleotidesequence identity to SEQ. ID. Nos. 1-26 or nucleic acid residues601-1000 of SEQ ID NO: 26 namely that exhibits at least 80% sequenceidentity, at least 85%, at least 90%, and at least 95%, 96%, 97%, 98% or99% sequence identity compared to SEQ. ID. Nos. 1-26 or residues601-1000 of SEQ ID NO: 26. Such sequence identity can be calculated bythe algorithms and computers programs described above.

The present invention further encompasses “functional variants” or“function fragments” of the disclosed sequences, particularly fragmentsof SEQ ID NOs: 1-26 and residues 601-1000 of SEQ ID NO: 5 that retainpromoter activity. Functional variants include, for example, regulatorysequences of the invention having one or more nucleotide substitutions,deletions or insertions and wherein the variant retains promoteractivity. Functional variants can be created by any of a number ofmethods available to one skilled in the art, such as by site-directedmutagenesis, induced mutation, identified as allelic variants, cleavingthrough use of restriction enzymes, or the like. Activity can likewisebe measured by any variety of techniques, including measurement ofreporter activity as is described at U.S. Pat. No. 6,844,484, Northernblot analysis, or similar techniques. The '484 patent describes theidentification of functional variants of different promoters.

Functional fragment, that is, a regulatory sequence fragment can beformed by one or more deletions from a larger regulatory element. Forexample, in some instances, the 5′ portion of a promoter up to the TATAbox near the transcription start site can be deleted without abolishingpromoter activity, as described by Opsahl-Sorteberg, H-G. et al.,“Identification of a 49-bp fragment of the HvLTP2 promoter directingaleruone cell specific expression” Gene 341:49-58 (2004). Such fragmentsshould retain promoter activity, particularly the ability to driveexpression of operably linked nucleotide sequences. Activity can bemeasured by Northern blot analysis, reporter activity measurements whenusing transcriptional fusions, and the like. See, for example, Sambrooket al., Molecular Cloning, A laboratory Manual (1989). Functionalfragments can be obtained by use of restriction enzymes to cleave thenaturally occurring regulatory element nucleotide sequences disclosedherein; by synthesizing a nucleotide sequence from the naturallyoccurring DNA sequence; or can be obtained through the use of PCRtechnology. See particularly, Mullis et al., Methods Enzymol,155:335-350 (1987) and Erlich, ed., PCR Technology (Stockton Press, NewYork), (1989).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′,4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

5. Testing of Promoters

Promoters of the invention, including functional fragments, are testedfor activity by cloning the sequence into an appropriate vector,transforming plants with the construct and assaying for marker geneexpression. Recombinant DNA constructs are prepared which comprise thepromoter sequences of the invention inserted into a vector suitable fortransformation of plant cells. The construct can be made using standardrecombinant DNA techniques (Sambrook et al. 1989) and can be introducedto the species of interest by Agrobacterium-mediated transformation orby other means of transformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89:    8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA 93:    9975-9979;-   (b) YAC: Burke et al. (1987) Science 236:806-812;-   (c) PAC: Sternberg N. et al. (1990) Proc Natl Acad Sci USA    87(1):103-7;-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al. (1995) Nucl    Acids Res 23: 4850-4856;-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al. (1983) J. Mol Biol 170: 827-842; or Insertion vector, e.g.,    Huynh et al. (1985) In: Glover N M (ed) DNA Cloning: A practical    Approach, Vol. 1 Oxford: IRL Press; T-DNA gene fusion vectors:    Walden et al. (1990) Mol Cell Biol 1: 175-194; and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a promotersequence of the present invention operationally linked to any markergene. The promoter was identified as a promoter by the expression of themarker gene. Although many marker genes can be used, Green FluorescentProtein (GFP) is preferred. The vector may also comprise a marker genethat confers a selectable phenotype on plant cells. The marker mayencode biocide resistance, particularly antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or phosphinotricin.Vectors can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

6. Constructing Promoters with Control Elements

6.1 Combining Promoters and Promoter Control Elements

The promoter and promoter control elements of the present invention,both naturally occurring and synthetic, can be used alone or combinedwith each other to produce the desired preferential transcription. Also,the promoters of the invention can be combined with other knownsequences to obtain other useful promoters to modulate, for example,tissue transcription specific or transcription specific to certainconditions. Such preferential transcription can be determined using thetechniques or assays described above.

Promoters can contain any number of control elements. For example, apromoter can contain multiple transcription binding sites or othercontrol elements. One element may confer tissue or organ specificity;another element may limit transcription to specific time periods, etc.Typically, promoters will contain at least a basal or core promoter asdescribed above. Any additional element can be included as desired. Forexample, a fragment comprising a basal or “core” promoter can be fusedwith another fragment with any number of additional control elements.

The following are promoters that are induced under stress conditions andcan be combined with those of the present invention: ldh1 (oxygenstress; tomato; see Germain and Ricard (1997) Plant Mol Biol 35:949-54),GPx and CAT (oxygen stress; mouse; see Franco et al. (1999) Free RadicBiol Med 27:1122-32), ci7 (cold stress; potato; see Kirch et al. (1997)Plant Mol Biol. 33:897-909), Bz2 (heavy metals; maize; see Marrs andWalbot (1997) Plant Physiol 113:93-102), HSP32 (hyperthermia; rat; seeRaju and Maines (1994) Biochim Biophys Acta 1217:273-80), and MAPKAPK-2(heat shock; Drosophila; see Larochelle and Suter (1995) Gene163:209-14).

In addition, the following examples of promoters are induced by thepresence or absence of light can be used in combination with those ofthe present invention: Topoisomerase II (pea; see Reddy et al. (1999)Plant Mol Biol 41:125-37), chalcone synthase (soybean; see Wingender etal. (1989) Mol Gen Genet 218:315-22) mdm2 gene (human tumor; see Saucedoet al. (1998) Cell Growth Differ 9:119-30), Clock and BMAL1 (rat; seeNamihira et al. (1999) Neurosci Lett 271:1-4, PHYA (Arabidopsis; seeCanton and Quail (1999) Plant Physiol 121:1207-16), PRB-1b (tobacco; seeSessa et al. (1995) Plant Mol Biol 28:537-47) and Ypr10 (common bean;see Walter et al. (1996) Eur J Biochem 239:281-93).

The promoters and control elements of the following genes can be used incombination with the present invention to confer tissue specificity:MipB (iceplant; Yamada et al. (1995) Plant Cell 7:1129-42) and SUCS(root nodules; broadbean; Kuster et al. (1993) Mol Plant MicrobeInteract 6:507-14) for roots, OsSUT1 (rice; Hirose et al. (1997) PlantCell Physiol 38:1389-96) for leaves, Msg (soybean; Stomvik et al. (1999)Plant Mol Biol 41:217-31) for siliques, cell (Arabidopsis; Shani et al.(1997) Plant Mol Biol 34(6):837-42) and ACT11 (Arabidopsis; Huang et al.(1997) Plant Mol Biol 33:125-39) for inflorescence.

Still other promoters are affected by hormones or participate inspecific physiological processes, which can be used in combination withthose of present invention. Some examples are the ACC synthase gene thatis induced differently by ethylene and brassinosteroids (mung bean; Yiet al. (1999) Plant Mol Biol 41:443-54), the TAPG1 gene that is activeduring abscission (tomato; Kalaitzis et al. (1995) Plant Mol Biol28:647-56), and the 1-aminocyclopropane-1-carboxylate synthase gene(carnation; Jones et al. (1995) Plant Mol Biol 28:505-12) and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod57:1467-77), both active during senescence.

Spacing between control elements or the configuration or controlelements can be determined or optimized to permit the desiredprotein-polynucleotide or polynucleotide interactions to occur.

For example, if two transcription factors bind to a promotersimultaneously or relatively close in time, the binding sites are spacedto allow each factor to bind without steric hindrance. The spacingbetween two such hybridizing control elements can be as small as aprofile of a protein bound to a control element. In some cases, twoprotein binding sites can be adjacent to each other when the proteinsbind at different times during the transcription process.

Further, when two control elements hybridize the spacing between suchelements will be sufficient to allow the promoter polynucleotide tohairpin or loop to permit the two elements to bind. The spacing betweentwo such hybridizing control elements can be as small as a t-RNA loop,to as large as 10 kb.

Typically, the spacing is no smaller than 5 bases; more typically, nosmaller than 8; more typically, no smaller than 15 bases; moretypically, no smaller than 20 bases; more typically, no smaller than 25bases; even more typically, no smaller than 30, 35, 40 or 50 bases.

Usually, the fragment size in no larger than 5 kb bases; more usually,no larger than 2 kb; more usually, no larger than 1 kb; more usually, nolarger than 800 bases; more usually, no larger than 500 bases; even moreusually, no more than 250, 200, 150 or 100 bases. In some embodiments,the nucleic acid of the invention comprises at least one fragment ofYP0286 (SEQ ID NO:5), e.g., YP2219 (SEQ ID NO:4), with the proviso thatsaid nucleic acid does not consist of YP0286 (SEQ ID NO:5).

Such spacing between promoter control elements can be determined usingthe techniques and assays described above.

6.2 Vectors Used to Transform Cells/Hosts

A plant transformation construct containing a promoter of the presentinvention may be introduced into plants by any plant transformationmethod. Methods and materials for transforming plants by introducing aplant expression construct into a plant genome in the practice of thisinvention can include any of the well-known and demonstrated methodsincluding electroporation (U.S. Pat. No. 5,384,253); microprojectilebombardment (U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208;6,399,861; and 6,403,865); Agrobacterium-mediated transformation (U.S.Pat. Nos. 5,824,877; 5,591,616; 5,981,840; and 6,384,301); andprotoplast transformation (U.S. Pat. No. 5,508,184).

The present promoters and/or promoter control elements may be deliveredto a system such as a cell by way of a vector. For the purposes of thisinvention, such delivery may range from simply introducing the promoteror promoter control element by itself randomly into a cell tointegration of a cloning vector containing the present promoter orpromoter control element. Thus, a vector need not be limited to a DNAmolecule such as a plasmid, cosmid or bacterial phage that has thecapability of replicating autonomously in a host cell. All other mannerof delivery of the promoters and promoter control elements of theinvention are envisioned. The various T-DNA vector types are a preferredvector for use with the present invention. Many useful vectors arecommercially available.

It may also be useful to attach a marker sequence to the presentpromoter and promoter control element in order to determine activity ofsuch sequences. Marker sequences typically include genes that provideantibiotic resistance, such as tetracycline resistance, hygromycinresistance or ampicillin resistance, or provide herbicide resistance.Specific selectable marker genes may be used to confer resistance toherbicides such as glyphosate, glufosinate or broxynil (Comai et al.(1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al. (1988) Science 242: 419-423). Other markergenes exist which provide hormone responsiveness.

The promoter or promoter control element of the present invention may beoperably linked to a polynucleotide to be transcribed. In this manner,the promoter or promoter control element may modify transcription bymodulating transcript levels of that polynucleotide when inserted into agenome.

However, prior to insertion into a genome, the promoter or promotercontrol element need not be linked, operably or otherwise, to apolynucleotide to be transcribed. For example, the promoter or promotercontrol element may be inserted alone into the genome in front of apolynucleotide already present in the genome. In this manner, thepromoter or promoter control element may modulate the transcription of apolynucleotide that was already present in the genome. Thispolynucleotide may be native to the genome or inserted at an earliertime.

Alternatively, the promoter or promoter control element may be insertedinto a genome alone to modulate transcription. See, for example,Vaucheret, H et al. (1998) Plant J 16: 651-659. Rather, the promoter orpromoter control element may be simply inserted into a genome ormaintained extrachromosomally as a way to divert transcription resourcesof the system to itself. This approach may be used to downregulate thetranscript levels of a group of polynucleotide(s).

The nature of the polynucleotide to be transcribed is not limited.Specifically, the polynucleotide may include sequences that will haveactivity as RNA as well as sequences that result in a polypeptideproduct. These sequences may include, but are not limited to antisensesequences, RNAi sequences, ribozyme sequences, spliceosomes, amino acidcoding sequences, and fragments thereof. Specific coding sequences mayinclude, but are not limited to endogenous proteins or fragmentsthereof, or heterologous proteins including marker genes or fragmentsthereof.

Constructs of the present invention would typically contain a promoteroperably linked to a transcribable nucleic acid molecule operably linkedto a 3′ transcription termination nucleic acid molecule. In addition,constructs may include but are not limited to additional regulatorynucleic acid molecules from the 3′-untranslated region (3′ UTR) of plantgenes (e.g., a 3′ UTR to increase mRNA stability of the mRNA, such asthe PI-II termination region of potato or the octopine or nopalinesynthase 3′ termination regions). Constructs may include but are notlimited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acidmolecule which can play an important role in translation initiation andcan also be a genetic component in a plant expression construct. Forexample, non-translated 5′ leader nucleic acid molecules derived fromheat shock protein genes have been demonstrated to enhance geneexpression in plants (see for example, U.S. Pat. Nos. 5,659,122 and5,362,865, all of which are hereby incorporated by reference). Theseadditional upstream and downstream regulatory nucleic acid molecules maybe derived from a source that is native or heterologous with respect tothe other elements present on the promoter construct.

Thus, one embodiment of the invention is a promoter such as provided inSEQ ID NOs: 1-26 or residues 601-1000 of SEQ ID NO: 26, operably linkedto a transcribable nucleic acid molecule so as to direct transcriptionof said transcribable nucleic acid molecule at a desired level or in adesired tissue or developmental pattern upon introduction of saidconstruct into a plant cell. In some cases, the transcribable nucleicacid molecule comprises a protein-coding region of a gene, and thepromoter provides for transcription of a functional mRNA molecule thatis translated and expressed as a protein product. Constructs may also beconstructed for transcription of antisense RNA molecules or othersimilar inhibitory RNA in order to inhibit expression of a specific RNAmolecule of interest in a target host cell.

Exemplary transcribable nucleic acid molecules for incorporation intoconstructs of the present invention include, for example, nucleic acidmolecules or genes from a species other than the target gene species, oreven genes that originate with or are present in the same species, butare incorporated into recipient cells by genetic engineering methodsrather than classical reproduction or breeding techniques. Exogenousgene or genetic element is intended to refer to any gene or nucleic acidmolecule that is introduced into a recipient cell. The type of nucleicacid molecule included in the exogenous nucleic acid molecule caninclude a nucleic acid molecule that is already present in the plantcell, a nucleic acid molecule from another plant, a nucleic acidmolecule from a different organism, or a nucleic acid molecule generatedexternally, such as a nucleic acid molecule containing an antisensemessage of a gene, or a nucleic acid molecule encoding an artificial ormodified version of a gene.

The promoters of the present invention can be incorporated into aconstruct using marker genes as described, and tested in transientanalyses that provide an indication of gene expression in stable plantsystems. As used herein the term “marker gene” refers to anytranscribable nucleic acid molecule whose expression can be screened foror scored in some way. Methods of testing for marker gene expression intransient assays are known to those of skill in the art. Transientexpression of marker genes has been reported using a variety of plants,tissues, plant cell(s), and DNA delivery systems. For example, types oftransient analyses can include but are not limited to direct genedelivery via electroporation or particle bombardment of tissues in anytransient plant assay using any plant species of interest. Suchtransient systems would include, but are not limited to, electroporationof protoplasts from a variety of tissue sources or particle bombardmentof specific tissues of interest. The present invention encompasses theuse of any transient expression system to evaluate promoters or promoterfragments operably linked to any transcribable nucleic acid molecules,including but not limited to selected reporter genes, marker genes, orgenes of agronomic interest. Examples of plant tissues envisioned totest in transients via an appropriate delivery system would include, butare not limited to, leaf base tissues, callus, cotyledons, roots,endosperm, embryos, floral tissue, pollen, and epidermal tissue.

Promoters and control elements of the present invention are useful formodulating metabolic or catabolic processes. Such processes include, butare not limited to, secondary product metabolism, amino acid synthesis,seed protein storage, increased biomass, oil development, pest defenseand nitrogen usage. Some examples of genes, transcripts and peptides orpolypeptides participating in these processes, which can be modulated bythe present invention: are tryptophan decarboxylase (tdc) andstrictosidine synthase (str1), dihydrodipicolinate synthase (DHDPS) andaspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma-zeins,ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus thuringiensis(Bt) insecticidal protein, cowpea trypsin inhibitor (CpTI), asparaginesynthetase and nitrite reductase. Alternatively, expression constructscan be used to inhibit expression of these peptides and polypeptides byincorporating the promoters in constructs for antisense use,co-suppression use or for the production of dominant negative mutations.

As explained above, several types of regulatory elements existconcerning transcription regulation. Each of these regulatory elementsmay be combined with the present vector if desired. Translation ofeukaryotic mRNA is often initiated at the codon that encodes the firstmethionine. Thus, when constructing a recombinant polynucleotideaccording to the present invention for expressing a protein product, itis preferable to ensure that the linkage between the 3′ portion,preferably including the TATA box, of the promoter and thepolynucleotide to be transcribed, or a functional derivative thereof,does not contain any intervening codons which are capable of encoding amethionine.

The vector of the present invention may contain additional components.For example, an origin of replication allows for replication of thevector in a host cell. Additionally, homologous sequences flanking aspecific sequence allow for specific recombination of the specificsequence at a desired location in the target genome. T-DNA sequencesalso allow for insertion of a specific sequence randomly into a targetgenome.

The vector may also be provided with a plurality of restriction sitesfor insertion of a polynucleotide to be transcribed as well as thepromoter and/or promoter control elements of the present invention. Thevector may additionally contain selectable marker genes. The vector mayalso contain a transcriptional and translational initiation region, anda transcriptional and translational termination region functional in thehost cell. The termination region may be native with the transcriptionalinitiation region, may be native with the polynucleotide to betranscribed, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al.(1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the polynucleotide to be transcribed may be optimizedfor increased expression in a certain host cell. For example, thepolynucleotide can be synthesized using preferred codons for improvedtranscription and translation. See U.S. Pat. Nos. 5,380,831, 5,436,391;see also and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

Additional sequence modifications include elimination of sequencesencoding spurious polyadenylation signals, exon intron splice sitesignals, transposon-like repeats, and other such sequences wellcharacterized as deleterious to expression. The G-C content of thepolynucleotide may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. The polynucleotide sequence may be modified to avoid hairpinsecondary mRNA structures.

A general description of expression vectors and reporter genes can befound in Gruber, et al. (1993) “Vectors for Plant Transformation” InMethods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 89-119, CRC Press. Moreover GUS expression vectors and GUS genecassettes are available from Clonetech Laboratories, Inc., Palo Alto,Calif. while luciferase expression vectors and luciferase gene cassettesare available from Promega Corp. (Madison, Wis.). GFP vectors areavailable from Aurora Biosciences.

6.3 Polynucleotide Insertion Into A Host Cell

The promoters according to the present invention can be inserted into ahost cell. A host cell includes but is not limited to a plant,mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.

The method of insertion into the host cell genome is chosen based onconvenience. For example, the insertion into the host cell genome mayeither be accomplished by vectors that integrate into the host cellgenome or by vectors which exist independent of the host cell genome.

The promoters of the present invention can exist autonomously orindependent of the host cell genome. Vectors of these types are known inthe art and include, for example, certain type of non-integrating viralvectors, autonomously replicating plasmids, artificial chromosomes, andthe like.

Additionally, in some cases transient expression of a promoter may bedesired.

The promoter sequences, promoter control elements or vectors of thepresent invention may be transformed into host cells. Thesetransformations may be into protoplasts or intact tissues or isolatedcells. Preferably expression vectors are introduced into intact tissue.General methods of culturing plant tissues are provided for example byMaki et al. (1993) “Procedures for Introducing Foreign DNA into Plants”In Methods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 67-88 CRC Press; and by Phillips et al. (1988) “Cell-Tissue Cultureand In-Vitro Manipulation” In Corn & Corn Improvement, 3rd EditionSprague et al. eds., pp.345-387, American Society of Agronomy Inc. etal.

Methods of introducing polynucleotides into plant tissue include thedirect infection or co-cultivation of plant cell with Agrobacteriumtumefaciens, Horsch et al. (1985) Science, 227:1229. Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer provided by Gruber et al. supra.

Alternatively, polynucleotides are introduced into plant cells or otherplant tissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably polynucleotides are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al., “Direct DNA transfer into intactplant cells via microprojectile bombardment” In: Gamborg and Phillips(Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods,Springer Verlag, Berlin (1995).

Methods for specifically transforming dicots are well known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut(Arachis hypogaea), and members of the genus Brassica.

Methods for transforming monocots are well known to those skilled in theart. Transformation and plant regeneration using these methods have beendescribed for a number of crops including, but not limited to, barley(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass(Dactylis glomerata); rice (Oryza sativa, including indica and japonicavarieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tallfescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostisstolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticumaestivum), switchgrass (Panicum vigatum) and alfalfa (Medicago sativa).It is apparent to those of skill in the art that a number oftransformation methodologies can be used and modified for production ofstable transgenic plants from any number of target plants of interest.

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp. or hybrids thereof, Sorghum spp.or hybrids thereof, sudangrass, Miscanthus spp. or hybrids thereof,Saccharum spp. or hybrids thereof, Erianthus spp., Populus spp.,Andropogon gerardii (big bluestem), Pennisetum purpureum (elephantgrass) or hybrids thereof (e.g., Pennisetum purpureum×Pennisetumtyphoidum), Phalaris arundinacea (reed canarygrass), Cynodon dactylon(bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata(prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giantreed) or hybrids thereof, Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale (Triticum—wheat×rye),Tripsicum dactyloides (Eastern gammagrass), Leymus cinereus (basinwildrye), Leymus condensatus (giant wildrye), and bamboo.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species such as, but not limited to, Sorghum almum,Sorghum ampium, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor(such as bicolor, guinea, caudatum, kafir, and durra), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum×almum, Sorghum×sudangrass or Sorghum×drummondii.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava).

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp.(ryegrass) and Phleum pratense (timothy).

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp.×Miscanthus sp., Panicum virgatum×Panicum amarum, Panicumvirgatum×Panicum amarulum, and Pennisetum purpureum×Pennisetumtyphoidum).

In another embodiment of the current invention, expression constructscan be used for gene expression in callus culture for the purpose ofexpressing marker genes encoding peptides or polypeptides that allowidentification of transformed plants. Here, a promoter that isoperatively linked to a polynucleotide to be transcribed is transformedinto plant cells and the transformed tissue is then placed oncallus-inducing media. If the transformation is conducted with leafdiscs, for example, callus will initiate along the cut edges. Oncecallus growth has initiated, callus cells can be transferred to callusshoot-inducing or callus root-inducing media. Gene expression will occurin the callus cells developing on the appropriate media: callusroot-inducing promoters will be activated on callus root-inducing media,etc. Examples of such peptides or polypeptides useful as transformationmarkers include, but are not limited to barstar, glyphosate,chloramphenicol acetyltransferase (CAT), kanamycin, spectinomycin,streptomycin or other antibiotic resistance enzymes, green fluorescentprotein (GFP), and β-glucuronidase (GUS), etc. Some of the promotersprovided in SEQ ID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ IDNO: 26 will also be capable of sustaining expression in some tissues ororgans after the initiation or completion of regeneration. Examples ofthese tissues or organs are somatic embryos, cotyledon, hypocotyl,epicotyl, leaf, stems, roots, flowers and seed.

Integration into the host cell genome also can be accomplished bymethods known in the art, for example, by the homologous sequences orT-DNA discussed above or using the cre-lox system (A. C. Vergunst et al.(1998) Plant Mol. Biol. 38:393).

7. Uses of the Promoters of the Invention

7.1 Use of the Promoters to Study and Screen for Expression

The promoters of the present invention can be used to further understanddevelopmental mechanisms. For example, promoters that are specificallyinduced during callus formation, somatic embryo formation, shootformation or root formation can be used to explore the effects ofoverexpression, repression or ectopic expression of target genes, or forisolation of trans-acting factors.

The vectors of the invention can be used not only for expression ofcoding regions but may also be used in exon-trap cloning, or promotertrap procedures to detect differential gene expression in varioustissues (see Lindsey et al. (1993) Transgenic Research 2:3347. Auch andReth (1990) Nucleic Acids Research 18: 6743).

Entrapment vectors, first described for use in bacteria (Casadaban andCohen (1979) Proc. Nat. Aca. Sci. U.S.A. 76: 4530; Casadaban et al.(1980) J. Bacteriol. 143: 971) permit selection of insertional eventsthat lie within coding sequences. Entrapment vectors can be introducedinto pluripotent ES cells in culture and then passed into the germlinevia chimeras (Gossler et al. aaa91989) Science 244: 463; Skarnes (1990)Biotechnology 8: 827). Promoter or gene trap vectors often contain areporter gene, e.g., lacZ, lacking its own promoter and/or spliceacceptor sequence upstream. That is, promoter gene traps contain areporter gene with a splice site but no promoter. If the vector lands ina gene and is spliced into the gene product, then the reporter gene isexpressed.

Recently, the isolation of preferentially-induced genes has been madepossible with the use of sophisticated promoter traps (e.g. IVET) thatare based on conditional auxotrophy complementation or drug resistance.In one IVET approach, various bacterial genome fragments are placed infront of a necessary metabolic gene coupled to a reporter gene. The DNAconstructs are inserted into a bacterial strain otherwise lacking themetabolic gene, and the resulting bacteria are used to infect the hostorganism. Only bacteria expressing the metabolic gene survive in thehost organism; consequently, inactive constructs can be eliminated byharvesting only bacteria that survive for some minimum period in thehost. At the same time, broadly active constructs can be eliminated byscreening only bacteria that do not express the reporter gene underlaboratory conditions. The bacteria selected by such a method containconstructs that are selectively induced only during infection of thehost. The IVET approach can be modified for use in plants to identifygenes induced in either the bacteria or the plant cells upon pathogeninfection or root colonization. For information on IVET see the articlesby Mahan et al. (1993) Science 259:686-688, Mahan et al. (1995) Proc.Natl. Acad. Sci. USA 92:669-673, Heithoff et al. (1997) Proc. Natl.Acad. Sci USA 94:934-939, and Wanget al. (1996) Proc. Natl. Acad. SciUSA 93:10434.

7.2 Use of the Promoters to Transcribe Genes of Interest

In one embodiment of the invention, a nucleic acid molecule as shown inSEQ ID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ NO: 26 isincorporated into a construct such that a promoter of the presentinvention is operably linked to a transcribable nucleic acid moleculethat is a gene of agronomic interest. As used herein, the term “gene ofagronomic interest” refers to a transcribable nucleic acid molecule thatincludes but is not limited to a gene that provides a desirablecharacteristic associated with plant morphology, physiology, growth anddevelopment, yield, nutritional enhancement, disease or pest resistance,or environmental or chemical tolerance. The expression of a gene ofagronomic interest is desirable in order to confer an agronomicallyimportant trait. A gene of agronomic interest that provides a beneficialagronomic trait to crop plants may be, for example, including, but notlimited to genetic elements comprising herbicide resistance, increasedyield, increased biomass, insect control, fungal disease resistance,virus resistance, nematode resistance, bacterial disease resistance,starch production, modified oils production, high oil production,modified fatty acid content, high protein production, fruit ripening,enhanced animal and human nutrition, biopolymers, environmental stressresistance, pharmaceutical peptides, improved processing traits,improved digestibility, industrial enzyme production, improved flavor,nitrogen fixation, hybrid seed production, and biofuel production. Thegenetic elements, methods, and transgenes described in the patentslisted above are hereby incorporated by reference.

Alternatively, a transcribable nucleic acid molecule can effect theabove mentioned phenotypes by encoding a RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product. Thus, anynucleic acid molecule that encodes a protein or mRNA that expresses aphenotype or morphology change of interest may be useful for thepractice of the present invention.

7.3. Stress Induced Preferential Transcription

Promoters and control elements providing modulation of transcriptionunder oxidative, drought, oxygen, wound, and methyl jasmonate stress areparticularly useful for producing host cells or organisms that are moreresistant to biotic and abiotic stresses. In a plant, for example,modulation of genes, transcripts, and/or polypeptides in response tooxidative stress can protect cells against damage caused by oxidativeagents, such as hydrogen peroxide and other free radicals.

Drought induction of genes, transcripts, and/or polypeptides are usefulto increase the viability of a plant, for example, when water is alimiting factor. In contrast, genes, transcripts, and/or polypeptidesinduced during oxygen stress can help the flood tolerance of a plant.

The promoters and control elements of the present invention can modulatestresses similar to those described in, for example, stress conditionsare VuPLD1 (drought stress; Cowpea; see Pham-Thi et al. (1999) Plant MolBiol 39:1257-65), pyruvate decarboxylase (oxygen stress; rice; seeRivosal et al. (1997) Plant Physiol 114(3): 1021-29), chromoplastspecific carotenoid gene (oxidative stress; Capsicum; see Bouvier et al.(1998) J Biol Chem 273: 30651-59).

Promoters and control elements providing preferential transcriptionduring wounding or induced by methyl jasmonate can produce a defenseresponse in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptides undersuch conditions is useful to induce a defense response to mechanicalwounding, pest or pathogen attack or treatment with certain chemicals.

Promoters and control elements of the present invention also can triggera response similar to those described for cf9 (viral pathogen; tomato;see O'Donnell et al. (1998) Plant J 14(1): 137-42), hepatocyte growthfactor activator inhibitor type 1 (HAI-1), which enhances tissueregeneration (tissue injury; human; Koono et al. (1999) J HistochemCytochem 47: 673-82), copper amine oxidase (CuAO), induced duringontogenesis and wound healing (wounding; chick-pea; Rea et al. (1998)FEBS Lett 437: 177-82), proteinase inhibitor II (wounding; potato; seePena-Cortes et al. (1988) Planta 174: 84-89), protease inhibitor II(methyl jasmonate; tomato; see Farmer and Ryan (1990) Proc Natl Acad SciUSA 87: 7713-7716), two vegetative storage protein genes VspA and VspB(wounding, jasmonic acid, and water deficit; soybean; see Mason andMullet (1990) Plant Cell 2: 569-579).

Up-regulation and transcription down-regulation are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease oxidative, flood, or drought tolerance may requireup-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in wounding or under methyl jasmonate induction, producetranscript levels that are statistically significant as compared to celltypes, organs or tissues under other conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.4. Light Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by light exposure can be utilized to modulate growth,metabolism, and development; to increase drought tolerance; and decreasedamage from light stress for host cells or organisms. In a plant, forexample, modulation of genes, transcripts, and/or polypeptides inresponse to light is useful

-   -   (1) to increase the photosynthetic rate;    -   (2) to increase storage of certain molecules in leaves or green        parts only, e.g. silage with high protein or starch content;    -   (3) to modulate production of exogenous compositions in green        tissue, e.g. certain feed enzymes;    -   (4) to induce growth or development, such as fruit development        and maturity, during extended exposure to light;    -   (5) to modulate guard cells to control the size of stomata in        leaves to prevent water loss, or    -   (6) to induce accumulation of beta-carotene to help plants cope        with light induced stress.

The promoters and control elements of the present invention also cantrigger responses similar to those described in: abscisic acidinsensitive3 (ABI3) (dark-grown Arabidopsis seedlings, see Rohde et al.(2000) Plant Cell 12: 35-52), asparagine synthetase (pea root nodules,see Tsai and Coruzzi (1990) EMBO J 9: 323-32), mdm2 gene (human tumor,see Saucedo et al. (1998) Cell Growth Differ 9: 119-30).

Up-regulation and transcription down-regulation are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease drought or light tolerance may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues or organs exposed to light, producetranscript levels that are statistically significant as compared tocells, tissues, or organs under decreased light exposure (intensity orlength of time).

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.5. Dark Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by dark or decreased light intensity or decreased light exposuretime can be utilized to time growth, metabolism, and development, tomodulate photosynthesis capabilities for host cells or organisms. In aplant, for example, modulation of genes, transcripts, and/orpolypeptides in response to dark is useful, for example,

-   -   (1) to induce growth or development, such as fruit development        and maturity, despite lack of light;    -   (2) to modulate genes, transcripts, and/or polypeptide active at        night or on cloudy days; or    -   (3) to preserve the plastid ultra structure present at the onset        of darkness.

The present promoters and control elements can also trigger responsesimilar to those described in the section above.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth and development may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription under exposure to dark or decrease light intensity ordecrease exposure time, produce transcript levels that are statisticallysignificant.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.6. Leaf Preferential Transcription

Promoters and control elements providing preferential transcription in aleaf can modulate growth, metabolism, and development or modulate energyand nutrient utilization in host cells or organisms. In a plant, forexample, preferential modulation of genes, transcripts, and/orpolypeptide in a leaf, is useful, for example,

-   -   (1) to modulate leaf size, shape, and development;    -   (2) to modulate the number of leaves; or    -   (3) to modulate energy or nutrient usage in relation to other        organs and tissues

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a leaf, producetranscript levels that are statistically significant as compared toother cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.7. Root Preferential Transcription

Promoters and control elements providing preferential transcription in aroot can modulate growth, metabolism, development, nutrient uptake,nitrogen fixation, or modulate energy and nutrient utilization in hostcells or organisms. In a plant, for example, preferential modulation ofgenes, transcripts, and/or polypeptide in a root, is useful,

-   -   (1) to modulate root size, shape, and development;    -   (2) to modulate the number of roots, or root hairs;    -   (3) to modulate mineral, fertilizer, or water uptake;    -   (4) to modulate transport of nutrients; or    -   (4) to modulate energy or nutrient usage in relation to other        cells, organs and tissues.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs of a root, produce transcriptlevels that are statistically significant as compared to other cells,organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.8. Stem/Shoot Preferential Transcription

Promoters and control elements providing preferential transcription in astem or shoot can modulate growth, metabolism, and development ormodulate energy and nutrient utilization in host cells or organisms. Ina plant, for example, preferential modulation of genes, transcripts,and/or polypeptide in a stem or shoot, is useful, for example,

-   -   (1) to modulate stem/shoot size, shape, and development; or    -   (2) to modulate energy or nutrient usage in relation to other        organs and tissues

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a stem or shoot,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.9. Fruit and Seed Preferential Transcription

Promoters and control elements providing preferential transcription in asilique or fruit can time growth, development, or maturity; or modulatefertility; or modulate energy and nutrient utilization in host cells ororganisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a fruit, is useful

-   -   (1) to modulate fruit size, shape, development, and maturity;    -   (2) to modulate the number of fruit or seeds;    -   (3) to modulate seed shattering;    -   (4) to modulate components of seeds, such as, storage molecules,        starch, protein, oil, vitamins, anti-nutritional components,        such as phytic acid;    -   (5) to modulate seed and/or seedling vigor or viability;    -   (6) to incorporate exogenous compositions into a seed, such as        lysine rich proteins;    -   (7) to permit similar fruit maturity timing for early and late        blooming flowers; or    -   (8) to modulate energy or nutrient usage in relation to other        organs and tissues.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of siliques or fruits,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.10. Callus Preferential Transcription

Promoters and control elements providing preferential transcription in acallus can be useful to modulating transcription in dedifferentiatedhost cells. In a plant transformation, for example, preferentialmodulation of genes, transcripts, in callus is useful to modulatetranscription of a marker gene, which can facilitate selection of cellsthat are transformed with exogenous polynucleotides.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease marker gene detectability, for example, may requireup-regulation of transcription.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.11. Flower Specific Transcription

Promoters and control elements providing preferential transcription inflowers can modulate pigmentation; or modulate fertility in host cellsor organisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a flower, is useful,

-   -   (1) to modulate petal color; or    -   (2) to modulate the fertility of pistil and/or stamen.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease pigmentation, for example, may requireup-regulation of transcription

Typically, promoter or control elements, which provide preferentialtranscription in flowers, produce transcript levels that arestatistically significant as compared to other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.12. Immature Bud and Inflorescence Preferential Transcription

Promoters and control elements providing preferential transcription in aimmature bud or inflorescence can time growth, development, or maturity;or modulate fertility or viability in host cells or organisms. In aplant, for example, preferential modulation of genes, transcripts,and/or polypeptide in a immature bud and/or inflorescence, is useful,

-   -   (1) to modulate embryo development, size, and maturity;    -   (2) to modulate endosperm development, size, and composition;    -   (3) to modulate the number of seeds and fruits; or    -   (4) to modulate seed development and viability.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in immature buds and inflorescences, produce transcriptlevels that are statistically significant as compared to other celltypes, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.13. Senescence Preferential Transcription

Promoters and control elements providing preferential transcriptionduring senescence can be used to modulate cell degeneration, nutrientmobilization, and scavenging of free radicals in host cells ororganisms. Other types of responses that can be modulated include, forexample, senescence associated genes (SAG) that encode enzymes thoughtto be involved in cell degeneration and nutrient mobilization(Arabidopsis; see Hensel et al. (1993) Plant Cell 5: 553-64), and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod 57:1467-77), both induced during senescence.

In a plant, for example, preferential modulation of genes, transcripts,and/or polypeptides during senescence is useful to modulate fruitripening.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease scavenging of free radicals, for example, mayrequire up-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs during senescence, producetranscript levels that are statistically significant as compared toother conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.14. Germination Preferential Transcription

Promoters and control elements providing preferential transcription in agerminating seed can time growth, development, or maturity; or modulateviability in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptide in agerminating seed, is useful,

-   -   (1) to modulate the emergence of the hypocotyls, cotyledons and        radical; or    -   (2) to modulate shoot and primary root growth and development;

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in a germinating seed, produce transcript levels that arestatistically significant as compared to other cell types, organs ortissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

8. GFP Experimental Procedures and Results

Procedures

The polynucleotide sequences of the present invention were tested forpromoter activity using Green Fluorescent Protein (GFP) assays in thefollowing manner.

Approximately 1-3 kb of genomic sequence occurring immediately upstreamof the ATG translational start site of the gene of interest was isolatedusing appropriate primers tailed with BstXI restriction sites. StandardPCR reactions using these primers and genomic DNA were conducted. Theresulting product was isolated, cleaved with BstXI and cloned into theBstXI site of an appropriate vector, such as pNewBin4-HAP1-GFP (see FIG.1).

Agrobacterium-Mediated Transformation of Arabidopsis

Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija(WS) plants are transformed with Ti plasmids containing nucleic acidsequences to be expressed, as noted in the respective examples, in thesense orientation relative to the 35S promoter in a Ti plasmid. A Tiplasmid vector useful for these constructs, CRS 338, contains theCeres-constructed, plant selectable marker gene phosphinothricinacetyltransferase (PAT), which confers herbicide resistance totransformed plants.

Ten independently transformed events are typically selected andevaluated for their qualitative phenotype in the T₁ generation.

Preparation of Soil Mixture: 24 L Sunshine Mix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J.R. Peters, Inc., Allentown, Pa.), which are first added to 3gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/ml benzylaminopurineto 900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plant is in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

High-throughput Screening of T₁ Transgenic Plants: Seed is evenlydispersed into water-saturated soil in pots and placed into a dark 4° C.cooler for two nights to promote uniform germination. Pots are thenremoved from the cooler and covered with 55% shade cloth for 4-5 days.Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis,Paris, France) is sprayed on plants (3 ml FINALE® diluted into 48 oz.water) and repeated every 3-4 days until only transformants remain.

GFP Assay

Tissues are dissected by eye or under magnification using INOX 5 gradeforceps and placed on a slide with water and coversliped. An attempt ismade to record images of observed expression patterns at earliest andlatest stages of development of tissues listed below. Specific tissueswill be preceded with High (H), Medium (M), Low (L) designations.

Flower Pedicel, receptacle, nectary, sepal, petal, filament, anther,pollen, carpel, style, papillae, vascular, epidermis, stomata, trichomeSilique Stigma, style, carpel, septum, placentae, transmitting tissue,vascular, epidermis, stomata, abscission zone, ovule OvulePre-fertilization: inner integument, outer integument, embryo sac,funiculus, chalaza, micropyle, gametophyte Post-fertilization: zygote,inner integument, outer integument, seed coat, primordia, chalaza,micropyle, early endosperm, mature endosperm, embryo Embryo Suspensor,preglobular, globular, heart, torpedo, late mature, provascular,hypophysis, radicle, cotyledons, hypocotyl Stem Epidermis, cortex,vascular, xylem, phloem, pith, stomata, trichome Leaf Petiole,mesophyll, vascular, epidermis, trichome, primordia, stomata, stipule,margin

T1 Mature: These are the T1 plants resulting from independenttransformation events. These are screened between stage 6.50-6.90 (i.e.the plant is flowering and 50-90% of the flowers that the plant willmake have developed), which is 4-6 weeks of age. At this stage themature plant possesses flowers, siliques at all stages of development,and fully expanded leaves. The plants are initially imaged under UV witha Leica Confocal microscope to allow examination of the plants on aglobal level. If expression is present, they are re-imaged usingscanning laser confocal microscopy.

T2 Seedling: Progeny are collected from the T1 plants giving the sameexpression pattern and the progeny (T2) are sterilized and plated onagar-solidified medium containing M&S salts. In the event that there isno expression in the T1 plants, T2 seeds are planted from all lines. Theseedlings are grown in Percival incubators under continuous light at 22°C. for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, andthe shoot meristem region of individual seedlings were screened untiltwo seedlings were observed to have the same pattern. In general, thesame expression pattern was found in the first two seedlings. However,up to 6 seedlings were screened before “no expression pattern” wasrecorded. All constructs are screened as T2 seedlings even if they didnot have an expression pattern in the T1 generation.

T2 Mature: The T2 mature plants were screened in a similar manner to theT1 plants. The T2 seeds were planted in the greenhouse, exposed toselection and at least one plant screened to confirm the T1 expressionpattern. In instances where there were any subtle changes in expression,multiple plants were examined and the changes noted in the tables.

T3 Seedling: This was done similar to the T2 seedlings except that onlythe plants for which we are trying to confirm the pattern are planted.

Image Data:

Images are collected by scanning laser confocal microscopy. Scannedimages are taken as 2-D optical sections or 3-D images generated bystacking the 2-D optical sections collected in series. All scannedimages are saved as TIFF files by imaging software, edited in AdobePhotoshop, and labeled in Powerpoint specifying organ and specificexpressing tissues.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

What is claimed is:
 1. A vector construct comprising: (a) a firstnucleic acid molecule consisting of the nucleotide sequence of SEQ IDNO:22, wherein said first nucleic acid molecule functions as a promoter;and (b) a second nucleic acid molecule to be expressed, wherein saidfirst nucleic acid molecule and said second nucleic acid molecule areheterologous to each other and are operably linked.
 2. The vectorconstruct according to claim 1, wherein said second nucleic acidmolecule encodes a polypeptide.
 3. The vector construct according toclaim 2, wherein said second nucleic acid molecule is operably linked tosaid first nucleic acid molecule in the sense orientation.
 4. The vectorconstruct according to claim 3, wherein said second, nucleic acidmolecule is transcribed into an RNA molecule that expresses thepolypeptide encoded by said second nucleic acid molecule.
 5. The vectorconstruct according to claim 1, wherein said second nucleic acidmolecule is operably linked to said first nucleic acid molecule in theantisense orientation.
 6. The vector construct according to claim 5,wherein transcription of said second nucleic acid molecule produces anantisense molecule.
 7. The vector construct according to claim 1,wherein transcription of said second nucleic acid molecule produces anRNAi molecule against an endogenous gene.
 8. The vector constructaccording to claim 2, wherein said second nucleic acid molecule encodesa polypeptide of agronomic interest.
 9. A plant or plant cell comprisingthe vector construct according to claim
 1. 10. A plant or plant cellstably transformed with the vector construct according to claim
 1. 11. Atransgenic seed of the plant according to claim
 9. 12. A method ofdirecting transcription comprising combining, in an environment suitablefor transcription: (a) a first nucleic acid molecule consisting of thenucleotide sequence of SEQ ID NO:22,wherein said first nucleic acidmolecule functions as a promoter; and (b) a second nucleic acidmolecule; wherein said first nucleic acid molecule and said secondnucleic acid molecule are heterologous to each other and operablylinked, and transcribing said second nucleic acid molecule.
 13. A methodof expressing an exogenous coding region in a plant comprising: (a)transforming a plant cell with the vector of claim 1; (b) regenerating astably transformed plant from the transformed plant cell of step (a);and (c) selecting a plant containing a transformed plant cell, whereinexpression of the second nucleic acid molecule results in production ofa polypeptide encoded by said second transcribable nucleic acidmolecule.
 14. A method of altering the expression of a gene in a plantcomprising: (a) transforming a plant cell with the vector constructaccording to claim 1, and (b) regenerating a stably transformed plantfrom said transformed plant cell.
 15. A plant prepared according to themethod of claim
 13. 16. A transgenic seed from the plant according toclaim
 15. 17. A method of producing a transgenic plant, said methodcomprising: (a) transforming a plant cell with the vector according toclaim 1; and (b) growing a plant from said plant cell.
 18. The method ofclaim 17, wherein said second nucleic acid molecule comprises a nucleicacid sequence encoding a polypeptide.
 19. The method of claim 17,wherein said second nucleic acid molecule is operably linked to saidfirst nucleic acid molecule in the antisense orientation.
 20. The methodof claim 17, wherein transcription of said second nucleic acid moleculeproduces an RNAi molecule.
 21. A plant or plant cell comprising thevector construct according to claim
 1. 22. A plant or plant cell stablytransformed with the vector construct according to claim
 1. 23. Atransgenic seed of the plant according to claim
 21. 24. A method ofproducing a transgenic plant, said method comprising: (a) introducinginto a plant cell the vector according to claim 1; and (b) growing aplant from said plant cell, wherein the plant cell comprises the vector.